Pseudomonas aeruginosa (PA) is an opportunistic pathogen that induces a rapidly developing and destructive
disease of the cornea and is a global cause of visual impairment and blindness. It is also the most commonly
recovered causative organism in contact lens-related disease in developed countries. Of most concern,
continued emergence of antibiotic-resistant bacterial strains poses a serious challenge for effective disease
management and adjunctive treatments are required. Therefore, the long-term objective of the studies
proposed is to test the regulatory role of microRNAs (miRNAs), a newly recognized, important level of gene-
expression regulation, in bacterial keratitis, identify new therapeutic targets and provide alternative treatment
strategies. To support this goal, preliminary published studies showed that the miR-183/96/182 cluster, which
produces miR-183, miR-96 and miR-182, is expressed in the cornea and in innate immune cells, including
macrophages (Mf) and polymorphonuclear neutrophils (PMN), in both mouse and human. Inactivation of this
cluster in mice led to decreased pro-inflammatory chemotactic cytokines (e.g., MCP1, MIP2 and IL-1ß) in the
cornea and a decreased severity of PA-induced keratitis in miR-183/96/182 cluster knockout (ko) mice.
Consistent with reduced chemotactic cytokines in ko mice, PMN number was decreased early (1 day post
infection, dpi) in disease, and bacterial load increased. Yet later in disease (5 dpi) PMN number was similar in
both groups, but bacterial load was significantly decreased in the ko animals. Other preliminary data showed
that PMN from ko mice had enhanced phagocytic and killing abilities, consistent with in vivo data showing
reduction of bacterial load in the cornea, despite similar PMN number. Regarding potential treatment strategies,
a pilot experiment with prophylactic subconjunctival and topical application of anti-miRs in PA-infected wild-
type mice, provided information that down-regulation of miR-183/96/182 cluster function successfully
decreased the severity of PA keratitis. Therefore, to achieve our long-term objectives, the following aims are
proposed: Aim 1 will test that inactivation of the miR-183/96/182 cluster in corneal resident Mf (myeloid cells)
decreases production of pro-inflammatory chemotactic cytokines, specifically, MCP1, IL-1ß and
MIP2, contributing to a decreased infiltration of PMN and Mf to the infected cornea. Aim 2 will test that
restoration of miR-183/96/182 cluster expression in myeloid cells of ko mice by breeding them to myeloid
specific Cre transgenic animals, is sufficient to reverse the corneal response to PA infection in vivo, as well as
phagocytosis and intracellular killing by infiltrating cells. Aim 3 will test that DAP12 is a direct target of the miR-
183/96/182 cluster in PMN and Mf, and mediates its regulation of these cells. Aim 4 will test that local
knockdown of miR-183/96/182 cluster function in the cornea is therapeutic for PA-induced keratitis.
It is anticipated that these studies will reveal novel molecular mechanisms of miR-183/96/182 cluster regulation
of innate immune responses in bacterial keratitis and provide a new target for its treatment.